A High-Throughput Method for Identifying N-Ethyl-N-Nitrosourea (ENU)-Induced Point Mutations in Zebrafish

Abstract

Publisher Summary This chapter focuses on the high-throughput method for identifying n-ethyl-n-nitrosourea (ENU). Prior to screening for ENU-induced mutations in zebrafish, a library is generated consisting of cryopreserved sperm isolated from the F1 progeny of ENU-mutagenized males. Although it is possible to screen for mutations in fish that are kept alive, a cryopreserved library has several advantages. The zebrafish has become an important model system for vertebrate biology. Although forward genetic screens have uncovered the functions of many zebrafish genes, until recently no reliable, inexpensive, and high-throughput technology for targeted gene disruptions was developed. Zebrafish forward genetic screens have been—and continue to be—exceptionally productive. However, as the content of the zebrafish genome becomes available in the form of primary sequence information, it becomes increasingly evident that many essential genes have not been identified by this approach. Mutant phenotypes might be subtle or even undetectable in forward genetic screens because of the nature of the screen. For example, most genetic screens performed to date have focused on identifying phenotypes during the embryonic period while the embryo is still transparent and have therefore been easy to screen for morphological defects in the light microscope or following staining with tissue-specific markers.